After the completion of human genome sequencing, movements to utilize the obtained gene information for the medical field such as diagnosis have been activated. The next targets after the genome sequencing are gene expression profile analysis, analysis of single nucleotide substitution (Single Nucleotide Polymorphisms; SNPs) in genes, and the like. The expression levels of genes expressed under various conditions and genetic mutations have been analyzed, and the functions of genes and the relationships of genes to diseases or drug sensitivity are being revealed from the analysis, and the accumulated gene information is used for not only diagnosis of diseases but also the selection of treatment.
In particular, SNPs and mutations are important targets for genetic testing such as diagnosis of diseases or risk characterization, and used in various fields including diabetes, rheumatism, cancer, mental illness, and heart disease. As a method for detecting SNPs, various methods have been developed. Concrete examples of such methods include an invader method, a sniper method, a TaqMan PCR method, a hybridization probe method, an SNPIT method, a pyrominisequencing method, a denaturing high performance liquid chromatography (DHPLC) method, an MALDI-TOF/MS method, and a nanochip method, as a method for rapid and high-throughput analysis.
In the field of cancer, molecular targeted drugs which target a specific molecule in the living body and suppress its function have been actively developed. Detection of single nucleotide substitution is positioned as an important test in determining the application of molecular targeted drugs when selecting treatment. For example, it is recommended to conduct mutation analysis testing for an EGFR gene or a KRAS gene before the use of anticancer drugs. This is because the presence or absence of mutation causes different drug effects, and it is guided that a drug should be administered by considering mutation. Another objective is to select a patient with a risk of severe adverse effects rather than drug efficacy and with low dosage effects by examining the presence or absence of such mutation in advance. Because of those reasons, the detection of particularly single nucleotide substitution is positioned as an important test in the field of cancer.
However, in the detection of an acquired mutation such as cancer, since wild-type nucleic acid molecules derived from normal cells which are dominant in a specimen affect as background, mutation such as single nucleotide substitution cannot be detected by analysis techniques as described above in many cases. To solve this problem, a Scorpion-ARMS method and a PNA-LNA PCR Clamping method have been developed (Patent literature 1).
The Scorpion-ARMS method is a method of analyzing a product obtained by selectively amplifying a mutated molecule using a combination of a primer designed on the basis of a mutated sequence so that the mutation point is positioned close to the 3′ terminus of the primer and another primer, by a fluorescent detection method, a Scorpion method. The PNA-LNA PCR Clamping method is a method in which a wild-type molecule is selectively blocked with a clamp primer which is designed on the mutation point and is complementary to the wild-type sequence, and a mutated molecule is selectively amplified and detected using a mutated LNA as a fluorescent detection probe.
These methods utilize a difference in thermal stability in the equilibrium system of a hybrid which is formed from a primer or probe and a template molecule. The difference between hybrid formation when the primer or probe is completely complementary to the template molecule, and incomplete hybrid formation when the primer or probe is not complementary to the template molecule by one to several nucleotides is merely a difference in thermal stability. Therefore, appropriate conditions capable of distinguishing the wild-type molecule from the mutated molecule are different in accordance with the nucleotide sequence of interest, and conditions which change thermal stability equally act on both molecules even under appropriate conditions. That is to say, so long as only the difference in thermal stability of hybrid formation in the equilibrium system is utilized as the principle of the distinction between both, we have to select temperature conditions including compromise between the balance of specificity and sensitivity, and the range width of selectable temperature conditions is extremely narrow in many cases, and therefore, the design of probe and primer is often difficult for some gene sequences.
Under these circumstances of detection techniques, strict limitations are provided to collect specimens in current mutation detection testing for cancer. More particularly, it is recommended that pathological specimens are prepared from cancer tissues, the tumor site is identified from stained specimens, and only the tumor is collected from unstained specimens of serial sections (The Guidance on the measurement of KRAS gene mutations in colon cancer patients). However, the identification of the tumor site requires specialized knowledge of structural morphology, and its procedures are complicated and high cost.
Therefore, a detection method with high specificity and high sensitivity, in which there are a few limitations on the collection of specimens and the nucleotide sequence of a target gene as the requirements of testing, is desired.
As a detection method with high specificity and high sensitivity, a method for detecting a mutated gene utilizing a photo-crosslinking nucleic acid has been developed. For example, Patent literature 2 discloses a method for detecting a target nucleic acid having a specific nucleotide sequence, based on hybrid formation with a complementary chain, with high specificity and high sensitivity. In this method, a photo-crosslinking nucleic acid complementary to the target nucleic acid, and a photo-crosslinked nucleic acid having a base moiety capable of photo-crosslinking with the photo-crosslinking nucleic acid at the 3′ or 5′ terminus are used, and one of both nucleic acids has a label portion, and the other is immobilized on a substrate. According to this method, the photo-crosslinking nucleic acid and the photo-crosslinked nucleic acid on the same chain are specifically crosslinked with each other, utilizing photo-crosslinking, only when a complete hybrid is formed, the nucleic acid molecule with the label portion can be covalently immobilized on the substrate, complete washing can be performed under conditions where complementary double-stranded chains dissociate, and high specificity and high sensitivity are achieved.
However, the detection sensitivity in this method has a lower limit, and thus, this method can be used to detect the presence or absence of mutation contained in a large amount of target nucleic acid, but cannot be used to detect a small amount of mutated nucleic acid which coexists with a large amount of wild-type nucleic acid, because the content of the mutated nucleic acid in a small amount is less than or equal to the detection sensitivity in many cases. Further, since the photo-crosslinking nucleic acid is covalently bound to the photo-crosslinked nucleic acid on the same chain, the mutation contained in the target nucleic acid to be detected cannot be amplified.
On the other hand, Patent literature 3 discloses a method in which a sample containing a target nucleic acid is first subjected to amplification by PCR, and then the method disclosed in Patent literature 2 is performed, to detect a nucleic acid having one target nucleotide sequence or two or more target nucleotide sequences in the nucleic acid sample. When the content of the mutated nucleic acid is small, the content percentage of the wild-type nucleic acid does not change, even if the amplification by PCR can be performed, and thus, the mutated nucleic acid cannot be amplified to the detectable level by the amplification within the range detectable in vitro. Therefore, this method can be used to detect the presence or absence of mutation contained in a large amount of target nucleic acid, but cannot be used to detect a small amount of mutated nucleic acid which coexists with a large amount of wild-type nucleic acid. In addition, since a sample containing the target nucleic acid is subjected to amplification by PCR, if the content percentage of the mutated nucleic acid is higher than a certain level, there is a possibility to detect the mutated nucleic acid, but this method needs many steps and is complicated, and thus, cannot meet the demand of the clinical scene which requires rapid test results.